Identification of the biosynthetic units of Cypridina luciferin in Cypridina (Vargula) hilgendorfii by LC/ESI-TOF-MS

doi:10.1016/j.tet.2004.09.080

Tetrahedron

Volume 60, Issue 50, 6 December 2004, Pages 11427-11434

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Abstract

In a luminous ostracod Cypridina (Vargula) hilgendorfii, Cypridina luciferin with an imidazopyrazinone structure (3,7-dihydroimidazopyrazin-3-one) is utilized for the luminescence reaction. To identify the biosynthetic units of Cypridina luciferin, the stable isotope labeled compounds were examined by feeding experiments with living Cypridina specimens. The incorporation of the labeled compounds into Cypridina luciferin was identified by the method of LC/ESI-TOF-MS analyses and these results suggested that l-tryptophan, l-arginine and l-isoleucine are structural units of Cypridina luciferin.

With the feeding experiment of stable isotope labeled compounds in a luminous ostracod Cypridina (Vargula) hilgendorfii, l-tryptophan, l-arginine and l-isoleucine are identified as the structural units of Cypridina luciferin by LC/ESI-TOF-MS analysis.

Introduction

In bioluminescent marine organisms, Cypridina luciferin (1) and coelenterazine (2) having an imidazopyrazinone structure (3,7-dihydroimidazopyrazin-3-one, Fig. 1) are used for the luminescence reaction as follows; $Luciferin + O_{2} \overset{Luciferase}{⟶} Oxyluciferin + {CO}_{2} + Light$

Cypridina luciferin (1) is isolated from a luminous ostracod crustacean, Cypridina hilgendorfii (presently Vargula hilgendorfii), living near the Japanese coast.¹ It is also utilized for the luciferase reaction in certain kinds of bioluminescent fishes including Apogon, Parapriacanthus and Porichthys.² On the other hand, coelenterazine (2) is widely distributed in luminous and non-luminous coelenterates, fishes, shrimps and squids,³ and is known as Watasenia preluciferin, Renilla luciferin and Oplophorus luciferin.⁴ Further, 2 serves as the chromogenic compound of photoproteins including aequorin and obelin.⁵

The luminescence system of Cypridina has been investigated extensively, since Harvey reported the luciferin-luciferase reaction with extracts of the specimens in 1917.⁶ When Cypridina specimens are stimulated physically or electronically, it expels Cypridina luciferin (1) and luciferase directly into the seawater to produce a brilliant bluish luminescence (λ_max=460 nm). After the luminescent reaction, 1 is converted to oxyluciferin (3), which is then hydrolyzed to etioluciferin (4) (Fig. 2).⁷ The chemical mechanism of the luminescence reaction catalyzed by Cypridina luciferase was proposed.⁸ Its luciferase gene was isolated⁹ and has been utilized as a reporter protein.¹⁰ The isolation, structural determination and total synthesis of Cypridina luciferin (1) was achieved¹ and Kishi et al. proposed that 1 should be biosynthesized from three amino acids or their equivalents: l-arginine, l-isoleucine and l-tryptophan (or tryptamine).¹

Recently, we reported that l-tryptophan is one of the structural units of Cypridina luciferin (1) in its biosynthesis.¹¹ After feeding of the deuterium labeled l-tryptophan as bait to Cypridina specimens for 6 days, the ethanol extract from living specimens was analyzed by LC/ESI-TOF-MS and the incorporation of the deuterium labeled l-tryptophan into Cypridina luciferin was identified. In this study, other possible amino acids for the biosynthetic units of Cypridina luciferin (1) were examined and we concluded that natural amino acids of l-arginine, l-isoleucine and l-tryptophan are structural units, but not d-tryptophan and tryptamine.

Section snippets

ESI-TOF-MS analyses of natural and synthetic Cypridina luciferin (1)

Before identifying the incorporation of stable isotopic compounds into Cypridina luciferin (1) by LC/ESI-TOF-MS, 1 was analyzed by the infusion method with ESI-TOF-MS. As the authentic compounds, chemically synthesized dl-Cypridina luciferin (1) and natural 1 were used and the peaks of the monovalent and divalent ions were detected in the positive mode (Table 1 and Fig. 3). The mass value of monovalent ion corresponding to Cypridina luciferin (1) was mainly observed at m/z 405.2 as [M]⁺, but

Conclusion

After feeding experiments of the stable isotope labeled compounds to C. hilgendorfii, Cypridina luciferin (1) extracted from the living specimens was analyzed by LC/ESI-TOF-MS. From the mass spectral analysis, we concluded that Cypridina luciferin (1) is biosynthesized from three amino acids, l-tryptophan, l-arginine and l-isoleucine in C. hilgendorfii.

Materials

[¹³C₆,¹⁵N]-l-Isoleucine (9)(l-[U–¹³C₆,U–¹⁵N; >98%]isoleucine; chemical purity >95%) was purchased from Spectra Stable Isotopes (Columbia, MD, USA). [¹⁵N₂]-l-Tryptophan (l-[U–¹⁵N₂; 96–99%]tryptophan, chemical purity >98%), [¹³C₆,¹⁵N₄]-l-arginine (8)(l-[U–¹³C₆; >99%, U–¹⁵N₄; >99%]arginine), [¹³C₆]-l-Isoleucine (10)(l-[U–¹³C₆; >98%]isoleucine, chemical purity >98%) and D₂O (99.9 atom % D) were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). DCl (20 wt% solution, 99.5 atom%

Acknowledgements

We thank Drs. H. Michibata and T. Ueki of Mukaishima Marine Biological Laboratory, Hiroshima University in Japan for assistance in collection of animals. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (A) from the Ministry of Education, Culture, Sports, Science and Technology and a JSPS fellowship for S. Kato.

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Identification of the biosynthetic units of Cypridina luciferin in Cypridina (Vargula) hilgendorfii by LC/ESI-TOF-MS

Abstract

Introduction

Section snippets

ESI-TOF-MS analyses of natural and synthetic Cypridina luciferin (1)

Conclusion

Materials

Acknowledgements

Comp. Biochem. Physiol.

Comp. Biochem. Physiol.

Mar. Biol.

J. Biolumi. Chemilumi.

Am. J. Physiol.

Science

Acc. Chem. Res.

Proc. R. Soc. Edinb., Ser. B

Tetrahedron Lett.

Bull. Chem. Soc. Jpn

Tetrahedron Lett.

Tetrahedron Lett.

Science

Proc. Natl. Acad. Sci. U.S.A.

Biochem. Biophys. Res. Commun.

Chem. Lett.

Tetrahedron Lett.

Proc. Natl Acad. Sci. U.S.A.

Chem. Commun.